Method of culturing segmented filamentous bacteria in vitro

ABSTRACT

The present invention relates to an in vitro method of culturing a segmented filamentous bacterium strain, comprising co-culturing said segmented filamentous bacterium strain with a eukaryotic host cell, wherein the culture is performed at an O 2  level inferior to 5% in a rich tissue culture liquid medium containing bacterial medium components including iron. The present invention also relates to methods for genetically modifying a segmented filamentous bacterium strain comprising a step a culturing the strain in vitro.

The present invention relates to an in vitro method for growingsegmented filamentous bacteria (SFB). The present invention also relatesto methods for genetically modifying a SFB strain.

Segmented filamentous bacteria (SFB) or Candidatus arthromitus orCandidatus savagella or Arthromitus immunis or Arthromitus muris aregram-positive anaerobic, clostridia-related, spore-forming commensalsfound in the gut (distal illeum) of many vertebrate species, includingmouse, rat, rabbit, birds (e.g., chicken), fish, amphibian and probablyhumans (Klaasen et al., 1992; Yin et al., 2012). The size of thefilament is comprised between 0.5 and 1000 μm in length. SFB produceintracellular offsprings (Schnupf et al., 2013; Ericsson et. al., 2014for review). Intracellular offsprings once formed are unicellular untilthey start to filament and become bicellular, then tricellular.

SFB have garnered much interest because of their unique ability toeducate the gut immune system and to induce a healthy state ofphysiological inflammation (Schnupf et al., 2013; Ivanov et al., 2009).SFB colonization leads to the maturation of the gut mucosal lymphoidtissue, induces a strong and broad IgA response, stimulates the T-cellcompartment, upregulates intestinal innate defence mediators, and inducea striking increase in the small-intestinal Th17 responses (Schnupf etal., 2013; Ivanov et al., 2009; Gaboriau-Routhiau et al., 2009). Inaddition, SFB colonization exerts an adjuvant effect on systemicresponses and can thus exacerbate pathologies in mouse models ofencephalitis and arthritis, while protecting genetically predisposedmice against the development of type I diabetes (Lee et al., 2011; Wu etal., 2010; Chappert et al., 2013; Kriegel et al., 2011; Yurkovetskiy etal., 2013).

Unlike other commensals, the segmented filamentous bacterium intimatelyattaches to absorptive epithelial cells in the ileum and cells overlyingPeyer's patches (Jepson et al., 1993; Chase et al., 1976). Thiscolonization does not result in pathology; rather, it protects the hostfrom pathogens (Ivanov et al., 2009).

The genomes of several SFB strains are known in the art. Recentsequencing of the rat and mouse SFB genomes revealed the highlyauxotrophic needs of SFB and placed SFB between obligate and facultativesymbionts (Pamp et al., 2012). These findings suggest that SFB obtain atleast some of their nutritional requirements from their interaction withthe host (Prakash et al., 2011; Sczesnak et al., 2011; Kuwahara et al.,2011; Pamp et al., 2012).

Yet, little is known about the segmented filamentous bacterium-hostinteraction that underlies the important immunostimulatory properties ofthe bacterium, because SFB have resisted in vitro culturing for morethan 50 years.

Therefore, there is a need for an effective method of culturing SFB invitro.

The inventors have grown mouse SFB outside their host in a segmentedfilamentous bacterium-host cell co-culturing system. The inventors haveshown that in this system, single-celled SFB isolated from monocolonizedmice undergo filamentation, segmentation, and differentiation to releaseviable infectious particles, the unicellular intracellular offspring,which can colonize mice to induce signature immune responses. Theinventors have also observed in this co-culturing system the formationof SFB spores. In vitro, intracellular offspring can attach to mouse andhuman host cells and recruit actin. Further, SFB can potently stimulatethe upregulation of host innate defence genes, inflammatory cytokines,and chemokines. These immunostimulatory properties of SFB increase hostresistance to pathogens and can be exploited to direct immunity toparticular pathogens of interest.

Accordingly, the present invention provides an in vitro method ofculturing a segmented filamentous bacterium (SFB) strain, comprisingco-culturing said segmented filamentous bacterium strain with aeukaryotic host cell, preferably mammalian host cell, more preferablyhuman host cell, wherein the culture is performed at an O₂ levelinferior to 5%, preferably inferior to 4%, in a rich tissue cultureliquid medium containing bacterial medium components including iron,preferably at a concentration between 0.015 and 0.05 mM.

As used herein a segmented filamentous bacterium strain refers to aCandidatus arthromitus or Candidatus savagella or Arthromitus immunis orArthromitus muris strain, preferably a Arthromitus muris strain.

Advantageously, the method comprises the steps of:

a) growing a eukaryotic host cell on a solid culture medium;

b) immerging the eukaryotic host cells grown in step a) in a eukaryotichost cell-SFB liquid culture medium;

c) challenging the cell culture of step b) with a SFB strain, preferablyan intracellular SFB offspring;

d) co-culturing the live eukaryotic host cell and the SFB strain in theliquid culture medium at an O₂ level inferior to 5%, preferably inferiorto 4%; and optionally

e) recovering the cultured SFB strain.

With Regard to Step a):

The eukaryotic host cell can be any vertebrate cell, preferably anymammalian cell, more preferably any human or murine cell (e.g., a mousecell).

In a preferred embodiment the eukaryotic host cell is an adherent cellin vitro. Advantageously, this adherent cell is able to differentiateand hence fares well under confluent conditions for extended periods oftime in vitro.

In another preferred embodiment, the eukaryotic host cell is anepithelial cell. Advantageously, the eukaryotic host cell is a cell fromthe gastrointestinal tract, such as the colon or small intestine.

In another embodiment, the eukaryotic host cell is a cancer cell,preferably a carcinoma cell, more preferably an adenocarcinoma cell.

In a most preferred embodiment, the eukaryotic host cell is a mammaliangastrointestinal epithelial adherent cell or a mammalian adherent cancercell, in particular a human or mouse colon epithelial or small intestinecell or a human or mouse adenocarcinoma cell.

In a particular embodiment, the eukaryotic host cell is selected fromthe group consisting of the human Caco-2, TC7 (which is a subclone ofCaco-2) or HeLa cell lines or mouse mICcl2 or CMT93 cell lines,preferably the human Caco-2 or TC7 cell line.

Advantageously, the eukaryotic host cell is grown at an O₂ level between0% (anaerobic conditions) and 5% (i.e., between 0 and 37.1 Torr),preferably between 0.5% and 3% (i.e., between 3.7 and 22.3 Torr), morepreferably between 1 and 2.5% (i.e., between 7.4 and 18.6 Torr).

The humidity and temperature conditions for growing the eukaryotic hostcell can be determined by routine tests by a person skilled in the art.The temperature is preferably between 36° C. and 38° C., more preferably37 degrees Celcius.

The composition of the eukaryotic host cell culture medium can bedetermined by routine tests by a person skilled in the art. By way ofexample, the culture medium comprises Dulbecco's Modified Eagle Medium(DMEM) or Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12(DMEM/F12) advanced medium, inactivated (or decomplemented) fetal calfserum (FCS) and amino acids.

Advantageously, the eukaryotic host cell is grown until a cellconfluence of at least 20%, and by order of increasing preference of atleast 25%, 30%, 35%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 98%, and more preferably of 100%, is obtained.

In a preferred embodiment, the eukaryotic host cell is grown until acell monolayer is obtained.

In another preferred embodiment, the eukaryotic host cells grown in stepa) are plated on a plate, preferably on a tissue culture well ortranswell, more preferably on a tissue culture transwell, prior to stepb).

Advantageously, the seeding density of the eukaryotic host cell isbetween 1×10⁴ and 6×10⁴ cells per cm². By way of example the seedingdensity of the eukaryotic host cell is between 6×10⁴ and 12×10⁴ cellsper culture well (i.e. per 3.8 cm²) or 3×10⁴ and 6×10⁴ cells per culturetranswell (i.e. per 1.12 cm²).

Advantageously, when the SFB are added in step c), the culture densityof the eukaryotic host cell is between 0.5×10⁵ and 3×10⁵ cells per cm².By way of example the culture density of the eukaryotic host cell isbetween 3×10⁵ and 6×10⁵ cells per culture well (i.e. per 3.8 cm²) or1×10⁵ and 3×10⁵ cells per culture transwell (i.e. per 1.12 cm²).

With Regard to Step b):

The eukaryotic host cell-SFB liquid culture medium is a medium suitablefor culturing both the eukaryotic host cell and the SFB strain.

Advantageously, said liquid culture medium is a rich tissue culturemedium (e.g., DMEM/F12 advanced medium) containing bacterial mediumcomponents including iron.

Said liquid culture medium can comprise brain-heart infusion and ayeast/peptone/casein amino-acid mixture. These bacterial mediumcomplements are well known in the art.

Iron can be in the ferrous form (Fe²⁺ state) and/or ferric (Fe³⁺ state).By way of example, iron can be in the form of Fe²⁺/Fe³⁺, 3×Fe²⁺, 3×Fe²⁺or hemin, preferably Fe²⁺/Fe³⁺, 3×Fe²⁺ or 3×Fe²⁺.

Advantageously, iron concentration is between 0.015 and 0.05 mM,preferably between 0.02 and 0.04 mM.

In a preferred embodiment, said liquid culture medium comprises a mediumfor culturing the eukaryotic host cell, such as DMEM/F12 advancedmedium, supplemented with fetal calf serum, non-essential amino acids(such as Glutamax™), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid(HEPES), brain-heart infusion, a yeast/peptone/casein amino-acid mixtureand iron.

The liquid culture medium can be supplemented with decomplemented fetalcalf serum (FCS), in particular when the liquid culture medium comprisesDMEM or DMEM/F12 advanced medium. Advantageously, the liquid medium issupplemented with 1% to 5% decomplemented FCS, preferably between 1% to3%, more preferably 2%.

Advantageously, said liquid culture medium further comprises sugars,retinoic acid and/or nucleotides.

With Regard to Step c):

The SFB strain can be a wild-type strain or a genetically modifiedstrain. A genetically modified SFB strain can be obtained from awild-type SFB strain by transformation, transduction or conjugation.

The SFB strain can be in the form of a filament or an intracellularoffspring.

The SFB strain can be isolated from a subject, in particular a mammal,such as a human or a mouse (e.g., a SFB monoassociated mouse). Methodsfor isolating a SFB strain from a mammal are known in the art (Lécuyeret al., 2014).

An individual SFB filament can be isolated by the following method: theileal, caecal, and/or colonic contents from a subject is collected andthe individual filament is captured using an inverted microscope and amicropipette attached to a micromanipulator.

An intracellular SFB offspring can be isolated under anaerobic or lowaerobic conditions (i.e., between 0%-2% oxygen) by the following method:the ileal, caecal, and/or colonic contents from a subject is collected,filtered through a 100-μm mesh, separated from other faecal matter usinga density gradient column and passed through a 5-μm filter to obtain apure culture of unicellular intracellular offspring (average 0.7 μm).SFB filaments can also be collected on 5 μm filters following thismethod.

As used herein, the term “challenging” refers to the addition of abacterium to an eukaryotic host cell regardless of whether thisbacterium interacts (attaches/invades) or not the host cell.

In a preferred embodiment, the SFB strain is closely contacted with theeukaryotic host cell, preferably is in direct contact with theeukaryotic host cell.

As used herein, the term “closely contacted” refers to a co-culture of aeukaryotic host cell and a SFB strain wherein the distance between saideukaryotic host cell and said SFB strain is inferior or equal to 2 cm.

Advantageously, the seeding density of the SFB is between 0.2×10⁴ and10.10⁴ cells per cm². By way of example the seeding density of the SFBis between 1.10⁴ and 10.10⁴ bacteria per culture well (i.e. per 3.8 cm²)or 1.10⁴ and 10.10⁴ bacteria per culture transwell (i.e. per 1.12 cm²).

Advantageously, the ratio between the eukaryotic host cells and the SFBis between 0.1 and 100, preferably between 0.3 and 60.

With Regard to Step d):

The co-culture of the eukaryotic host cell and the SFB is performed atan O₂ level between 0% (anaerobic conditions) and 5%, preferably between0.5 and 3%, more preferably between 1 and 2.5%.

The humidity and temperature conditions for co-culturing the liveeukaryotic host cell and the SFB strain can be determined by routinetests by a person skilled in the art. The temperature is preferablybetween 36° C. and 38° C., more preferably 37° C.

The co-culture can be performed for 1, 2, 3, 4, 5 or 6 days.

The co-culture can also be performed until the SFB strain undergoesfilamentation, segmentation, differentiation to release intracellularoffsprings or spores.

Conditions to induce spore formation advantageously include adding astress such as oxidative stress or short times of antibiotics (Davis andSavage, 1976).

With Regard to Step e)

The cultured SFB strain can be recovered in a form of a filament, anintracellular offspring or a spore.

A SFB filament can be recovered using an inverted microscope and amicropipette attached to a micromanipulator.

A SFB intracellular offspring can be recovered from a filament byfiltration through a 5-μm filter. SFB filaments can be collected on 5 μmfilters.

A SFB spore (that is a bacterial spore) can be recovered by a methodwell known in the art, such as the methods disclosed in InternationalApplication WO 2014/121298.

The present invention also provides the Arthromitus muris straindeposited by the Applicant, according to the Budapest Treaty, at CNCM(Collection Nationale de Cultures de Microorganismes, 25 rue du DocteurRoux, Paris) on Dec. 23, 2014, under the accession number CNCM 1-4932.

The present invention also provides a method for genetically modifying asegmented filamentous bacterium strain by conjugation, comprising thesteps of:

i) genetically modifying an Escherichia coli strain with a recombinantshuttle vector DNA comprising a DNA sequence of interest to introduce inthe SFB strain, wherein the said shuttle vector DNA is capable ofreplicating in the E. coli strain, and in the SFB strain to be targetedfor genetic modification;

ii) co-culturing the genetically modified E. coli strain obtained instep i) together with the SFB strain to be targeted for geneticmodification according to the in vitro method of culturing of theinvention, wherein:

in step c) of said method of culturing of the invention, the cellculture of step b) is challenged with both the SFB strain to be targetedfor genetic modification, preferably an intracellular SFB offspring, andsaid genetically modified E. coli strain; and

in step d) of said method of culturing of the invention, the liveeukaryotic host cell, the SFB strain and the genetically modified E.coli strain are co-cultured in the liquid culture medium at an O₂ levelinferior to 5%;

iii) Recovering the Genetically Modified SFB Strain of Step ii).

The recombinant shuttle vector DNA can be a recombinant Clostridialshuttle vector DNA comprising a DNA sequence of interest and capable ofreplicating both in an E. coli strain and in a Clostridia strain. Theconstruction of such a vector is known in the art (Heap et al., 2009).

Advantageously, the DNA sequence of interest can be under the control ofa constitutive SFB, such as ribosomal, promoter for cytosolic expressionor fused to a nucleotide sequence encoding a secreted lipoprotein forsurface expression. By way of example, a strong promoter functional inSFB is the promoter of the SFB SFBNYU_003340 gene described by Yang etal. 2014.

The recombinant shuttle vector DNA can comprise one or differentselectable antibiotic markers (i.e., antibiotic resistance cassettes).Any selectable antibiotic marker can be used, such as lincomycin,spectinomycin, thiamphenicol, chloramphenicol and tetracycline,preferably lincomycin. In in vitro SFB growth inhibition tests, SFB issensitive to lincomycin at 0.1 ug/ml, spectinomycin at 4 ug/ml,thiamphenicol at 1 ug/ml, chloramphenicol at 5 ug/ml and tetracycline at0.6 ug/ml. Advantageously, the recombinant shuttle vector DNA comprisesa ERM/Lincomycin resistance cassette.

The DNA sequence of interest can be a DNA sequence encoding an antigenof interest. The DNA sequence encoding said antigen of interest isadvantageously codon optimized for expression in SFB, e.g., followingthe method described by Sczesnak et al. 2011). By way of example, theantigen of interest can be IpaB or IpaD from Shigella flexneri, intiminand heat labile toxin from EPEC and the colonization factor antigen I(Cfa/I) adhesin CfaE from ETEC.

An example of a DNA sequence of interest under the control of thepromoter of the SFB SFBNYU_003340 gene that has been codon optimized isprovided in SEQ ID NO: 17. It encodes a Green Fluorescent Protein(GFP)-Ovalbumine (Ova) peptide fusion protein.

The E. coli strain can be a negatively-selectable auxotrophic E. colistrain (Danchin, 1977).

Advantageously, the E. coli strain requires diaminopimelic acid to grow.Such a strain can be selected against diaminopimelic acid in a richculture medium.

The genetic modification of the E. coli strain can be carried out byelectroporation, transduction, heat shock transformation or protoplastfusion.

The co-culturing of step ii) allows transferring the said shuttle vectorDNA from the E. coli strain to the SFB by conjugation.

Advantageously, the cell culture of step b) is challenged first with theSFB strain to be targeted for genetic modification, and then with saidgenetically modified E. coli strain after there is outgrowth of the SFBstrain.

The present invention also provides a method for genetically modifying asegmented filamentous bacterium strain by electroporation, comprisingthe steps of:

i) culturing and recovering a segmented filamentous bacterium strainaccording to the in vitro method of the invention;

ii) mixing the SFB strain recovered in step i) with a purified plasmidto be transformed, preferably in glycerol/H₂O (e.g., 10% glycerol/H₂O);

iii) applying an electric pulse to the mixture obtained in step ii);

iv) recovering the genetically transformed SFB strain.

In step i) of this method, the SFB strain can be cultured for 2-4 dayson TC7 cells.

In step i) of this method, the SFB strain can be recovered by collectingthe SFB cell and putting it on ice under O₂ level between 0% (anaerobicconditions) and 5%, preferably between 0.5 and 3%, more preferablybetween 1 and 2.5%.

In step ii) of this method, the electric field is advantageously between6 kV.cm⁻¹ and 30 kV.cm⁻¹, preferably between 6.5 kV.cm⁻¹ and 25 kV.cm⁻¹.

In step ii) the purified plasmid can be designed for use in Clostridiaspecies (Heap et al., 2009). By way of example one can use the plasmidpMTL80110, pMTLP82254, pMTL83353, pMTL84422, pMTL85141, pMTL82151,pMTL83151, pMTL84151 or pMTL85151, all described by Heap et al., 2009(also referred in GENBANK database under these accession numbers).Advantageously, the plasmid is selected from the group consisting ofpMTL82151, pMTL83151, pMTL84151 and pMTL85151, further comprising anERM/Lincomycin resistance cassette cloned between the FseI site and thePmeI site of the multiple cloning site of these plasmids. These plasmidsare referred to as pMTL82151-ERM, pMTL83151-ERM, pMTL84151-ERM andpMTL85151-ERM, respectively described in SEQ ID NO: 18, 19, 20 and 21.

The plasmids can be purified by standard methods, mostly usingcommercially available kits such as Qiagen® plasmid purification kit forexample.

In step iv), if the plasmid comprises an antibiotic resistance cassettethen the SFB transformed with the plasmid is selected in vitro with thecorresponding antibiotic marker.

The present invention also provides a method for genetically modifying asegmented filamentous bacterium strain by chemical transformation,comprising the steps of:

i) culturing and recovering a segmented filamentous bacterium strainaccording to the in vitro method of the invention;

ii) mixing the SFB strain recovered in step i) with a purified plasmidto be transformed, preferably in glycerol/calcium chloride (e.g., 10%glycerol/0.1M calcium chloride);

iii) incubating the mixture obtained in step ii);

iv) optionally applying a heat shock to the mixture;

v) recovering the genetically transformed SFB strain.

In step i) of this method, the SFB strain can be cultured for 2-4 dayson TC7 cells.

In step i) of this method, the SFB strain can be recovered by collectingthe SFB cell and putting it on ice under O₂ level between 0% (anaerobiccondition) and 5%, preferably between 0.5 and 3%, more preferablybetween 1 and 2.5%.

In step iii), the SFB strain and the purified plasmid can be incubatedfor 15 to 45 min, preferably 30 min.

In step iv), the heat shocked is advantageously applied at 41-43° C.,preferably at 42° C. for 15 to 45 seconds, preferably 30 seconds.

In step v), if the plasmid comprises an antibiotic resistance cassettethen the SFB transformed with the plasmid is selected in vitro with thecorresponding antibiotic marker.

The methods for genetically modifying a segmented filamentous bacteriumstrain according to the invention can further comprise a step of in vivoselecting the transformed SFB strain comprising: administrating (e.g.,orally) the in vitro selected SFB transformed with a plasmid comprisingan antibiotic resistance cassette into the gut of a mouse, thenadministrating to said mouse the corresponding antibiotic marker andthen recovering the lived genetically transformed SFB strain from saidmouse. The antibiotic marker (e.g., lincomycin) can be added to thedrinking water of the mouse to be administered to said mouse. By way ofexample, lincomycin can be used in the drinking water at a low dilution,preferably between 50-500 μg/L.

A live genetically modified SFB strain can be used as an antigendelivery vehicle, either as filament, intracellular offspring or spore,preferably as a spore, after oral ingestion or parenteral administrationto a subject, preferably a mammal.

Accordingly, the present invention also provides an isolated geneticallymodified SFB strain expressing an antigen of interest (e.g., IpaB orIpaD from Shigella flexneri, intimin and heat labile toxin from EPEC andthe colonization factor antigen I (Cfa/I) adhesin CfaE from ETEC),preferably secreting said antigen or exposing said antigen onbacterium's surface.

The terms “a genetically modified SFB strain expressing an antigen ofinterest” and “a SFB strain genetically modified to express an antigenof interest” are used herein interchangeably.

As used herein, an antigen of interest refers to an (foreign) antigenderived from a microorganism or a cell (e.g., tumor cell) different froma SFB against which one intends to elicit an immune response in asubject. The microorganism can be a bacterium, virus or fungus organism.The cell is preferably of mammalian origin, preferably of human origin.

Given the potent ability of SFB to induce both B and T cell responses,including both Th1 and particularly Th17 cells, the antigen isadvantageously selected to elicit a Th17 response.

Particular embodiments of the antigen include antigens derived frommainly surface proteins of the diarrheal pathogens Shigella, enterotoxicEscherichia coli (ETEC) or attaching and effacing lesion (A/E)-inducingenteropathogenic E. coli (EPEC). For a Shigella antigen, one can choosethe type three secretion apparatus tip proteins IpaB and IpaD that arerequired for host cell invasion (Parsot 2009). IpaB and IpaD areconserved across different Shigella serotypes and, as recombinantproteins complexed with a double mutant heat-labile toxin, elicit a 90%protective efficacy to lethal pulmonary challenge of Shigella afterintranasal immunization and a 40% protective efficacy when administeredorally (Heine et al., 2013). In addition, IpaB-directed antibodies havebeen shown to inhibit Shigella plaque formation in vitro (Mills et al.,1988). For an ETEC antigen, one can choose the colonization factorantigen I (Cfa/I) adhesin CfaE that is located at the fimbrial tip andelicits protection from ETEC in a non-human primate Aotus nancymaaemodel after intradermal administration (Fleckenstein et al., 2014). AsCfa/I is only present in a subset of EPEC strains, it can be alsoinclude the heat-labile toxin (LT). The heat-labile toxin is highlyimmunogenic and a double-mutant of the toxin is used in vaccine researchas a mucosal adjuvant (Martinez-Becerra et al., 2012). However, as SFBalready has strong immunostimulatory potential, only the nontoxic Bsubunit of LT (LTB) that can by itself elicit protective antibodyresponses to the toxin when expressed cytosolically or surface localizedby Bacillus subtilis can be selected (Paccez et al., 2007). For an EPECantigen, one can choose the adhesin intimin. Intimin mediates thecharacteristic formation of the attaching and effacing (A/E) lesions bybinding to the translocated receptor Tir (Kaper et al., 2004) and isfound in both typical and atypical EPEC, in enterohaemorrhagic E. coliand the murine EPEC-like strain C. rodentium. Intimin as a protectiveimmunogen has been validated in a number of animal models of infectionwith rabbit EPEC, EHEC and C. rodentium (Ferreira et al., 2011).Specifically it can be chosen a fragment encoding amino acids 363 to 808of the 94 kDa beta-intimin antigen, the most frequent EPEC intiminsubtype in human EPEC diarrheal cases and the subtype present in C.rodentium. This fragment can elicit protective immunity when given as asubcutaneous subunit vaccine or when delivered orally or sublingually byan intimin fragment-expressing Lactobacillus strain and shows promise ininducing intimin type-independent immunity (Ferreira et al., 2008 and2011, Ghaem-Maghami, M. et al., 2001).

The genetically modified SFB strain can be obtained by transformation,transduction or conjugation as described above, preferably by a methodfor genetically modifying a segmented filamentous bacterium strainaccording to the invention.

The present invention also provides a live genetically modified SFBstrain expressing an antigen of interest as defined above for use as amedicament, in particular for use in preventing or treating a diseasecaused by a microorganism from which said antigen derives, or a cancer.

Said SFB strain can be in the form of a filament, an intracellularoffspring or a spore, preferably a spore.

The present invention also provides a therapeutic immunogenic (orvaccine) composition comprising a live genetically modified SFB strainexpressing an antigen of interest as defined above.

Said SFB strain can be in the form of a filament, an intracellularoffspring or a spore, preferably a spore.

The terms “immunogenic composition” and “vaccine composition” are usedinterchangeably herein.

The present invention also provides an immunogenic composition asdescribed above for use in preventing or treating a disease caused by amicroorganism from which said antigen derives or a cancer.

The genetically modified SFB strain expressing an antigen of interestand the immunogenic composition comprising the said genetically modifiedSFB strain according to the present invention are particularly suitablefor eliciting in a subject antibodies directed to said antigen.

Advantageous said immunogenic composition comprises a pharmaceuticallyacceptable carrier.

As used herein, a “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. Suitable carriers aredescribed in the most recent edition of Remington's PharmaceuticalSciences, a standard reference text in the field.

The genetically modified SFB strain expressing an antigen of interestand the immunogenic composition comprising the said genetically modifiedSFB strain according to the present invention are administered to amammal subject, preferably a human, in an amount sufficient to preventor attenuate the severity, extent of duration of the infection by themicroorganism from which said antigen derives or a cancer.

The therapeutically effective amount varies depending on the subjectbeing treated, the age and general condition of the subject beingtreated, the capacity of the subject's immune response to synthesizeantibodies, the degree of protection desired, the severity of thecondition to be treated, and its mode of administration, among otherfactors. An appropriate effective amount can be readily determined byone of skill in the art. A therapeutically effective amount will fall ina relatively broad range that can be determined through routine trials.

Following an initial vaccination, the subject may receive one or twobooster injections at an appropriate interval determined by one of skillin the art.

Typically, the immunogenic composition is prepared as an injectable form(either a liquid solution or suspension) or as a solid form suitable forsolution or suspension in a liquid carrier prior to injection.

Once formulated, the immunogenic composition may be administeredparenterally or by mucosal routes, preferably orally.

The present invention also provides the use of a genetically modifiedSFB strain expressing an antigen of interest or an immunogeniccomposition as defined above for the manufacture of a medicament,preferably of a preventive or therapeutic vaccine against amicroorganism from which said antigen derives in a subject or a cancer.

The present invention also provides a method for preventing and/ortreating a infection caused by a microorganism from which said antigenderives or a cancer, comprising administering to a subject in needthereof a genetically modified SFB strain expressing an antigen ofinterest or an immunogenic composition as defined above, in an amounteffective to inhibit said microorganism infection of susceptible cellsso as to thereby prevent or treat the infection or to inhibit the growthof cancer cells so as to thereby prevent or treat cancer.

The term “treating” includes the administration of a geneticallymodified SFB strain expressing an antigen of interest or an immunogeniccomposition as defined above to a patient who has an infection, asymptom of infection or a cancer, with the purpose to cure, heal,alleviate, relieve, alter, remedy, ameliorate, improve, or affect theinfection and/or the symptoms of the infection, or the cancer.

The term “preventing” means that the progression of an infection isreduced and/or eliminated, or that the onset of an infection or canceris delayed or eliminated.

The present invention also provides the use of a SFB strain obtainableby the in vitro method of culturing of the invention, as a probiotic.

Said SFB strain can be alive or dead, preferably alive.

Said SFB strain can be in the form of a filament, an intracellularoffspring or a spore, preferably a spore.

The present invention also provides a food product (e.g., dairy product)comprising a SFB strain obtainable by the in vitro method of culturingof the invention.

Said SFB strain can be alive or dead, preferably alive.

Said SFB strain can be in the form of a filament, an intracellularoffspring or a spore, preferably a spore.

In addition to the preceding features, the invention further comprisesother features which will emerge from the following description, whichrefers to examples illustrating the present invention, as well as to theappended figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Growth and growth requirements of SFB in vitro. a, Gram stainand length of SFB before and after 5-μm filtration; b, qPCR of SFBgrowth on host cells at the indicated conditions; c, SEM images of4-day-old SFB filaments grown anaerobically on TC7 cells on transwells;d-i, SFB growth at low oxygen concentrations (d), on the indicated cellline seeded on transwells; e, on TC7 cells at indicated cell confluence;f, g, under various b, i, h, Statistical analysis using the two-tailedttest (*P=<0.05, **P=<0.01, ***P=<0.001).

FIG. 2: Differentiation of SFB from filaments to intracellular offspringduring in vitro growth. A, Schematic representation of an SFB filamenthighlighting stages of its growth and differentiation. Gram stain of SFBafter 4 days of growth on B, TC7 cells incubated at the indicatedcondition, and C, TC7 cells grown on transwells in low oxygen. Analysisof SFB grown on mICcl2 cells (D-G) and TC7 cells (H) on transwells at1-2.5% O2: D, qPCR quantification (mean±s.d. of triplicates); E, lengthof individual SFBs; F, Gram stain; and G, segment length analysis ofrepresentative 2-day-old SFB filaments. H, SEM of SFB after 4 days ofgrowth. B-H, Representative images and values from one of threeexperiments performed in triplicate with (D) means±s.d.

FIG. 3: Viability, colonization, and immunostimulatory potential of invitro-grown SFB. a, SFB length after growth on TC7 cells on transwells;b, quantification and c, Gram stain of SFB growth on TC7 cells ontranswells before and after a 3-day sub-culturing of the 5-μm filtrate;d-k, analysis of germ-free C57BL/6 mice gavaged with eitherfilament/intracellular offspring mix, intracellular offspring, or faecesof SFB-monoassociated mice (SFB Vivo); d, quantification ofileum-associated SFB; e, k, Host gene expression in the ileal laminapropria; representative flow cytometry plots and quantification of (f,g) B220+B cell and (i, j) CD45+CD3+CD4+ T cell frequencies of theindicated markers; h, faecal secretory IgA quantification by ELISA. a-k,Images and values are representatives from one of two experimentsperformed (a-c) in duplicate with means±s.d. or (d-k) performed withfour to six mice per group. Statistical analysis using the two-tailedt-test (***P=<0.001).

FIG. 4: SFB-host cell interaction and host response. SEM of SFB attachedto subconfluent (A) and confluent (B) mICcl2 cells after 2 days ofintracellular offspring challenge. C, Merge (M) of4′,6-diamidino-2-phenylindole (DAPI) (D) and actin (A) stain of mICcl2cells challenged with intracellular offspring for 2 days showingconfocal slices (s) and Z-projections. D, SEM of 3-day-old SFB filamentattached to TC7 cells. Host response using qPCR analysis of mICcl2 cells(E, F, H) and TC7 cells (G) 3 days after challenge with (E-G) SFB or (H)various MAMPs. NS, not significant; BDL, below detection limit. Imagesand values are representatives from one of two experiments performed intriplicate (A-D) or are cumulative values from four experimentsperformed in triplicate with box plots of 25-75% centiles with min/maxwhiskers (E-G) or means±s.e.m.; (H) with a two-tailed t-test statisticalanalysis (*P=<0.05, **P=<0.01, ***P=<0.001).

FIG. 5. Intestinal colonization of in vitro-grown SFB and host response.a, b, qPCR quantification (a) and SEM (b) of in vitro-grown SFB used forgavage. c, qPCR quantification of SFB in faecal samples. d, SEM of SFBattachment in vivo at 25 days after gavage of C57BL/6 mice with SFB Vivoor SFB Vitro. e, SEM of 21-day SFBcolonized germ-free C3H/HeN mouseileum showing vacant attachment sites. f, Host gene expression in theileal lamina propria in conventional or germ-free mice colonized witheither SFB or E. coli for 21 days. Images and values are representativesfrom one of two experiments (a-e) or are cumulative values from twoexperiments performed with a total of seven germ free, four SFB, five E.coli, and four conventional mice (f), showing box plots of 25-75%centiles with median and minimum/maximum whiskers. c, f, Two-tailedt-test statistical analysis (*P=<0.05, **P=<0.01).

EXAMPLE 1 Growth and Host Interaction of Mouse Segmented FilamentousBacteria in Vitro

Materials and Methods

Cell Culture and SFB-specific Culture Medium

TC7, CMT93, and HeLa cells were cultured in DMEM (Gibco 31885) with 10%inactivated fetal calf serum (FCS; AbCys CVFSVF00-0U) and non-essentialamino acids (Invitrogen 11140-035). mICcl2 were maintained in DMEM/F12advanced medium (Gibco 12634) with 2% inactivated FCS and Glutamax(Gibco 35050) and supplemented with 10 nM hEGF (Sigma E9644), 50 nMdexamethasone (Sigma D4902) and 1 nM triiodothyronine (Sigma T5516).HeLa cells were obtained from ATCC. All cell lines were tested formycoplasma every 2 weeks and always found to be negative. Cells wereplated either on regular 12-well tissue culture plates or on Costartranswell plates with 0.4-μm filters (Sigma CLS3460 and FisherScientific W2127P) from 1 to 3 days before the experiment such that amonolayer was present at the start of the experiment and added to a lowoxygen chamber overnight for slowly lowering oxygen concentrations inthe medium of the cells. At the start of SFB culturing, the TC7, CMT93,HeLa and mICcl2 medium is first replaced with (equilibrated: 1% oxygen)medium before SFB addition (directly to the TC7 cells).

SFB medium was made up as follows: DMEM/F12 advanced medium with 2% FCS,Glutamax and 12.5 mM HEPES (Sigma H0887) with the followingsupplementation: 1 in 100 dilution of (1) brain-heart infusion (BD Difco237500) 5× concentrated, (2) peptone/yeast (BBL Biosafe 211862) at 10%and casein amino acids at 5% (DIFCO 0320-01-1), (3)ribose/cellobiose/mannose (Sigma: R9629, C7252, M6020) at 200 mM; 1 in1,000 dilution of (1) ferrous sulphate (Merck 3965) at 10 mM, (2) ferricammonium citrate (LabGuard 0658) at 12.5 mM, (3) Hemin (Sigma 51280) at1.5 mM in 50% ethanol with 1.4 N NH4OH, (4) sodium ascorbate at 10 mgml⁻¹ with 1-phosphoascorbate at 500 mM (Sigma: A4034, 49752); 1 in10,000 dilution of retinoic acid (Sigma R2625) at 30 mg ml⁻¹ in DMSO; 1in 500 dilution of (1) sperm DNA (Life Technologies 15632-011) at 10 mgml⁻¹ digested for 1 h with 10 μl DNaseI (Roche 04 716 728 001) at 37° C.and heat inactivated at 75° C. for 30 min, (2) RNA at 10 mg ml⁻¹ (SigmaR6750) undigested. SFB medium specific medium supplements, except forthe nucleotides and hemin were prepared fresh every 2 weeks andotherwise stored at 4 or −20° C. for retinoic acid, and nucleotides.When mICcl2 cells were used, SFB medium was further supplemented withhEGF, dexamethasone, and triiodothyronine. Notably, additionalsupplementation with 0.2% yeast extract (BD 212750; 1/100 from 20%)further improves SFB growth and it is not necessary to add ascorbate,phosphoascorbate or hemin.

Purification of Intracellular Offspring from SFB-monoassociated Mice andInfection Protocol

All liquids used for the isolation of intracellular offspring werepre-equilibrated overnight in an anaerobic chamber set to 0% oxygen.SFB-monoassociated JH^(−/−) mice were killed aseptically in a tissueculture hood and then placed in an anaerobic cabinet for dissection. Theileal, caecal, and colonic contents were resuspended in 50 ml PBS andhomogenized by vortexing. Homogenates were passed through a 100-μm meshto remove large faecal debris. The filtrate was spun at 8,000 g for 5min to pellet bacteria and insoluble material, and the pellet wasresuspended in 3 ml PBS per mouse killed, layered onto 3 ml 50% and 2 ml30% Nycodenz (AbCYS 1002424) solution made with PBS in 15 ml Falcontubes and spun for 10 min at 4,000 g. SFB within the 30% fraction werecollected, diluted in PBS, and bacteria were pelleted for 10 min at8,500 g. Pellets were resuspended in 15 ml pre-equilibrated PBS bypipetting/vortexing and filtered through a 5-μm filter (Sigma Z612502).The filtrate was again centrifuged for 5 min at 8,000 g and the pelletwas resuspended in an appropriate amount of pre-equilibrated culturemedium. Usually one mouse was killed for every four 12-well plates used,and 50 μl of bacterial suspension was added to each well. To facilitateadhesion, cells challenged with SFB were sealed in ziplock bags withinthe cabinets, removed from the cabinet, and spun for 10 min at 300 g.

SFB are allowed to grow in the presence of eukaryotic cells for 3 daysduring which the oxygen concentration is increased incrementally from 1%on day 0 to 1.5% on day 1, to 2% on day 2, and to 2.5% on day 3.Subcultures can be prepared on day 3 by taking aliquots of the culturesupernatant and using this to challenge freshly prepared eukaryoticcells (prepared as described above on transwell filters and at lowoxygen conditions).

SFB Recovery, and Quantification and Analysis of SFB Growth

To recover SFB, the culture supernatant was collected and centrifugedfor 4 min at 8,000 g and the pellet was resuspended in 100 μl PBS, ofwhich 20 μl was spotted on glass slides for the Gram stain, 30 μl wasmixed with an equal volume of 50% glycerol and frozen at −80° C., andthe remaining 50 μl was used for DNA extraction. The DNA was isolatedwith a Qiagen stool kit (51504; without the use of the inhibitor tablet)and diluted 1 in 20. SFB growth was enumerated by qPCR analysis of the16S rRNA genes using the following primer pairs: the SFB specific F:5′-AGGAGGAGTCTGCGGCACATTAGC-3′ (SEQ ID NO: 1); and the universal R:5′-TCCCCACTGCTGCCTCCCGTAG-3′ (SEQ ID NO: 2). For qPCR, 6 μl of dilutedSFB DNA was mixed with 1.5 μl of a 4 mM primer mix and 7.5 μl of PowerSybrGreen Master mix (Applied 4368708) and run on an ABI 7900HT machinein a 384-well plate. Statistical analysis used a two-tailed Student'st-test (*P=<0.05, **P=<0.01, ***P=<0.001). SFB segment length analysisused ImageJ. For SEM analysis, SFB-containing supernatants were washedwith PBS and suctioned onto 0.1-μm filters (Watman 110405) and fixed in0.1 M cacodylate buffer containing 2.5% glutaraldehyde before beingprocessed. Cells for SEM were fixed in PHEM buffer (18.14 g PIPES, 6.5 gHEPES, 3.8 g EGTA, 0.99 g MgSO4 per litre with 10 M KOH to pH 7.0)containing 4% sucrose and 2.5% glutaraldehyde, and processed for SEM.For fluorescence, cells were fixed in PBS/3.7% PFA, permeabilized withPBS/0.1% Triton X-100, stained with DAPI and A568-phalloidin, and stacksof 0.4-μm slices were taken on a Leica SP5 confocal microscope.

Colonization of Germ Free Mice with In Vitro-grown SFB

This experiment was done independently twice with similar results; oneexperiment is shown. SFB grown in vitro for 3 days on TC7 and mICcl2cells on transwells were divided in equal parts and one half wasfiltered through a 5-μm filter to obtain a fraction containingintracellular offspring only. Bacteria were concentrated bycentrifugation to obtain 0.25 ml of bacteria in PBS per mouse. Thenumber of animals used followed availability of animals, isolators, andinput quantities. Randomization or blinding was otherwise not performed.Two groups of four 11-week-old C57BL/6 male and female mice maintainedat the germ-free facility were starved for one night, gavaged with 0.25ml 400 mM sodium bicarbonate, followed by 0.25 ml of in vitro-grown SFB.Age-matched control mice were colonized with in vivo-derived SFB asdescribed below. Faecal samples were collected for each mouse at varioustimes during a 3 week period, SFB DNA was extracted using a Qiagen stoolkit and quantified on the basis of qPCR analysis of 16S rDNA andcomparison with a SFB DNA sample of known SFB genome concentration asdetermined by Illumina sequencing. To monitor SFB associated with theileum, DNA was extracted from frozen ileal biopsies using the method inGodon et al., 1997.

Isolation and Staining of Lamina Propria Lymphocytes from C57BL/6 Mice

Age-matched germ-free B6 mice and mice colonized with either in vitro-or in vivo-grown SFB for 3 weeks were analysed for their innate andadaptive immune response as described in Ivanov et al., 2009. Briefly,after excision of Peyer's patches, the mouse small intestine was washedin PBS, and ileal samples were placed in RNAlater for RNA extraction,complementary DNA (cDNA) synthesis, and qPCR analysis using SYBR orTaqman technologies (Applied Biosystems) and a QuantStudio7 qPCRmachine. Values were normalized to TfrC.

Lamina propria lymphocytes (LPL) were prepared as previously describedby Gaboriau-Routhiau et al., 2009. The remaining small intestine wasincubated four times in 60 ml of PBS-3 mM EDTA (Sigma) for 10 min at 37°C., and digested in 60 ml of RPMI 1640 with 20% FCS (Gibco), 100 U ml⁻¹collagenase (Sigma), and 175 U ml⁻¹ DNase I (Sigma) for 40 min at 37° C.LPL were then purified on a 40-80% Percoll gradient run for 15 min at2,000 g and resuspended in DMEMGlutamax with 8% FCS, 1 mM HEPES, 0.02 mMfolic acid, 0.67 mM L-arginine, and 0.27 mM L-asparagine (all fromSigma).

Analysis of LPL for surface antigens and intracellular expression ofIL-17 and IL-10 was by flow cytometry as described 5. Briefly, LPL werestimulated for 4 h with 100 ng ml⁻¹ phorbol 12-myristate 13-acetate and1 μg ml⁻¹ ionomycin, in the presence of Brefeldin A (10 μg ml⁻¹) (allfrom Sigma). Cells used for surface analysis were left unstimulated. Forsurface staining, LPL were labelled for 20 min at 4° C. with a cocktailof the following antibodies: FITC-anti-GL7 (clone GL7), PerCP-anti-CD8a(clone 53-6.7), APC-H7-anti CD4 (clone GK1.5), AF647-anti-B220 (cloneRA3-6B/2) (all from BD Pharmingen), PE-anti-IgA (Southern Biotech), andeFluor450-anti-CD45 (clone 30-F11) and PECy7-anti-CD3 (clone 145-2C11)(both from eBioscience).

For intracellular cytokine staining, cells were further fixed in 2% PFAfor 20 min at room temperature (˜24° C.), and washed and stainedovernight at 4° C. with PE-anti-IL-17 (clone TC11-18H10) andAPC-anti-IL-10 (clone JES5-16E3) (BD Pharmingen) diluted in PBS-1%FCS-0.5% saponin (Sigma). Labelled cells were analysed with a FACSCantoII and FACSDiva software (BD Biosciences). Gates were set on livingcells after Aqua live/dead dye exclusion (Invitrogen).

For qPCR analysis, the median value of germ-free mice was calculated andused as the reference value of 1 for comparison of the median value ofthe test samples.

Colonization of Germ-free Mice with SFB from Faeces and E. coli

Germ-free male and female C3H/HeN mice were obtained from INRA (ANAXEMplatform) germ-free facilities. Eight- to nine-week-old germ-free micewere gavaged with 0.5 ml of either fresh anaerobic cultures of E. coliMG1655 or faecal homogenate from SFBmonoassociated mice (Ivanov et al.,2009). Colonization by SFB was monitored in faeces through bacterial DNAextraction and 16S rDNA amplification by qPCR using specific primerpairs for SFB. Values were normalized to Ccl25, a constitutivelyexpressed epithelial cell marker, and compared with the median valuefrom germ-free control mice. Germ-free and gnotobiotic mice weremaintained in plastic isolators and fed ad libitum on a commercial dietsterilized by γ-irradiation (40 kGy). The numbers of animals usedfollowed availability of animals, isolators, and were obtained from twoindependent experiments. Randomization or blinding was otherwise notperformed. Gnotobiotic mice were killed on day 21 after colonization inparallel with age-matched germ-free controls. All animal procedures wereperformed in accordance with French legislation and EEC regulations forthe care and use of laboratory animals, approved by the local ethicscommittee and authorized by the French Ministry of Research.

Host Response In Vitro to SFB Growth and MAMP Stimulation

Host response to SFB and MAMPs included pooled results from fourindependent experiments with three technical samples. Generally aminimum of triplicate biological replicates were used and increased iftrends were clear but significance not. After 3 days of in vitro growthof SFB on either mICcl2 or TC7 cells on transwells in SFB medium lackinghemin and sodium ascorbate, at 1-2.5% oxygen, cells were lysed and RNAwas extracted using a Nucleospin RNA kit (Macherey-Nagel). cDNA wassynthesized using RNA superscript II, oligo dT, RNaseout, and dNTPs(Invitrogen), and pPCR was performed on an ABI 7900HT and QuantStudio7(Life Technologies) qPCR machine using the protocol described by Schnupfet al., 2012. TaqMan assays were performed as suggested by the supplier.Values were normalized to B2M and Ct values for Reg3γ, Tnfα, and Fabp2were set to 41 in control cells owing to the lack of transcriptdetection. MAMP stimulation used the following agonists at the highestconcentrations recommended by the supplier (Invivogen): Pam2CSK4(tlrlpm2 s-1) at 100 ng ml⁻¹, Pam3CSK4 (tlrl-pms) at 300 ng ml⁻¹,peptidoglycan of E. coli K12 (Tlr-ksspgn) at 10 μg ml⁻¹, MDP (tlrl-mdp)at 10 μg ml⁻¹, CpG (ftr-1584) at 3 μg ml⁻¹, flagellin (tlrl-pstfla-5) at100 ng ml⁻¹. Excess for flagellin (10×) was also tested and found to besimilar.

Mouse and Human qPCR Primers Used

Mouse Taqman® Assays:

B2M Mm00437762_m1, TfrC Mm00441941_m1, Ccl25 Mm00436443_m1, Fabp2Mm00433188_m1, Reg3 g Mm01181783_g1, Fut2 Mm00490152_S1, Aqp3Mm01208559_m1, Pigr Mm00465049_m1, Ltf Mm00434787_m1, Tff3Mm00495590_m1, Tnfa Mm00443258_m1, IL1a Mm00439620_m1, IL1bMm00434228_m1, IL6 Mm00446190_m1, IL15 Mm00434210_m1, IL18Mm00434225_m1, Cxcl2 Mm00436450_m1, Ccl2 Mm00436450_m1, Ccl5Mm01302428_m1, Ccl7 Mm01308393_g1, Ccl20 Mm01268754_m1, Ccl28Mm00445039_m1, Csf2 Mm01290062_m1

Mouse Sybr® Primers

B2M:  (SEQ ID NO: 3) F: tcagtcgtcagcatggctcgc;  (SEQ ID NO: 4)R: tccggtgggtggcgtgagtatac  iNos:  (SEQ ID NO: 5)F: cagctgggctgtacaaacctt;  (SEQ ID NO: 6) R: cattggaagtgaagcgtttcg Saa1:  (SEQ ID NO: 7) F: catttgttcacgaggcatcc;  (SEQ ID NO: 8)R: gtttttccagttagcttccttcatgt  Saa2:  (SEQ ID NO: 9)F: tgtgtatcccacaaggtttcaga;  (SEQ ID NO: 10)R: ttattaccctctcctcctcaagca  Saa3:  (SEQ ID NO: 11)F: cgcagcacgagcaggat;  (SEQ ID NO: 12) R: ccaggatcaagatgcaaagaatg Cxcl1:  (SEQ ID NO: 13) F: tggctgggattcacctcaag;  (SEQ ID NO: 14)R: caagcctcgcgaccattct  Cxcl10:  (SEQ ID NO: 15)F: gccgtcattttctgcctcat;  (SEQ ID NO: 16) R: gcttccctatgcccctcatt 

Human Taqman® Assays:

B2M Hs00984230_m1, Saa1 Hs00761940_s1, Saa2 Hs01667582_m1, IL6Hs00985639_m1, IL8 Hs99999034_m1, Ccl20 Hs01011368_m1, Reg3 gHs00417999_m1, iNos Hs01075529_m1, Cxcl10 Hs00171042_m1, Fut2Hs00382834_m1, IL15 Hs01003716_m1, IL18 Hs01038788_m1, Ccl5Hs00982282_m1

Results

SFB-host Cell Co-culturing System

SFB strain (Bolotin et al., 2014) isolated from monoassociated mice wasdeposited at CNCM (Collection Nationale de Cultures de Microorganismes,25 rue du Docteur Roux, Paris) on Dec. 23, 2014, under the accessionnumber CNCM 1-4932 (Arthromitus muris strain referred to asSFB-mouse-NL). SFB were cultured with eukaryotic cells grown in low butphysiological oxygen conditions (He et al., 1999) in a rich tissueculture medium containing bacterial medium components and additionalsupplements. SFB from monoassociated mice were collected, filteredthrough a 100-μm mesh, separated from most other faecal matter using aNycodenz column and passed through a 5-μm filter to obtain a pureculture of unicellular intracellular offspring (average 0.7 μm) (FIG. 1a). Eukaryotic cells grown on tissue culture wells or transwells wereplaced in a humidified anaerobic cabinet, challenged with intracellularoffspring, and kept at either strict anaerobic conditions within asealed box or left in the anaerobic cabinet where the oxygenconcentration was maintained at low levels (0.5-1.4% O2). After 4 days,bacterial growth in the culture supernatant was quantified byquantitative PCR (qPCR) using SFB-specific 16S rDNA primers (FIG. 1b )and confirmed using scanning electron microscopy (SEM) (FIG. 1c ). SFBgrowth was observed in all conditions for most cell lines assayed butgrowth was usually enhanced on transwells compared with tissue culturewells and growth was significantly better in the presence of oxygen,revealing SFB to be a relatively aerotolerant anaerobe. Both human(TC7/HeLa) and mouse (mICcl2/CMT93) cell lines supported growth but theTC7 was the most resilient cell line generally supporting SFB growthmost robustly. On the basis of temporal analysis, the highestexponential growth phase occurred between days 1 and 3, with an averagemaximum doubling time of 5.0 h (FIG. 1d ).

Growth Requirements for SFB

SFB growth had a striking dependence on host cell number, decreasing innumber with decreasing cell density (FIG. 1e ). In addition, negligiblegrowth occurred in medium alone, medium supplemented with cell lysate,or when cells were fixed before SFB challenge (FIG. 1f ), indicatingthat live host cells are required for SFB proliferation. SFB alsorequired close contact for efficient growth as only little growth (0-6%)occurred when intracellular offspring were added to the bottom chamberof transwells or when bacteria were placed in transwells above TC7 cellsin tissue culture wells (FIG. 1f, g ). Yet, as host cell contact was notan absolute requirement, it suggests that host cells may release asoluble factor that promotes SFB growth. To address the requirement formedium supplementation, SFB were grown on TC7 cells in complete mediumor medium missing individual additives. Brain-heart infusion, ayeast/peptone/casein amino-acid mixture, and particularly ironsupplementation were critical for SFB growth (FIG. 1h, i ).

In the 1970s, transmission electron microscopy studies of SFB present inthe murine gut led to a proposed life cycle: attachment to epithelialcells via the holdfast at the intracellular offspring tip leads tofilamentous growth and is followed by a complex developmentalprogression that starts at the distal tip and ultimately leads tointracellular offspring formation and release (FIG. 2A) (Chase et al.,1976; Ferguson et al., 1979). According to this model, when filamentsgrow beyond 50 μm in length, the large primary filament segments startto undergo a symmetrical division to form smaller secondary segments.These differentiate by dividing asymmetrically to form a mother/daughtercell. The daughter cell becomes engulfed and subsequently divides toform two intracellular offspring within the surrounding mother cellsegment. Intracellular offspring are then released from the filament bybreakdown of the filament septa and cell wall and reattach to the host.

In vitro, growth of SFB on TC7 cells often yielded considerablequantities of long filaments that could be clumped together in ahairball-like phenotype easily seen with the naked eye (FIG. 2B). Mostof these filaments were undifferentiated after 4 days, with only somefilaments showing a characteristic heterogeneous staining ofdifferentiating filaments (FIG. 2B, left-hand column, bottom twopanels). Intracellular offspring could be seen located at the filamenttip or occasionally in the central part of a filament (FIG. 2C), similarto filaments recovered from SFB-monoassociated mice (data not shown).Differentiation of SFB occurred on all four cell lines tested, but wenoticed that differentiation was more pronounced when higher oxygenconcentrations (1-2.5%) were used. In time-course analysis at thishigher oxygen concentration (FIG. 2D), only short bacteria were detected1 day after challenge (FIG. 2E, F). After 2 days, only long filamentswere present (FIG. 2E, F) and the bacterial septa could be identifiedquite clearly using the Gram stain (FIG. 2F). Three types of filamentwere found (FIG. 2F, G): short filaments with long intracellularsegments of ˜2.6 μm; medium-sized filaments with smaller intracellularsegments of ˜1.2 μm; and medium to long filaments that had a moreheterogeneous distribution of segment lengths including very smallsegments (FIG. 2F, G) and rare segments with half-circular structures(FIG. 2F, Day 2-3, arrow) that resembled the engulfment of a daughtercell by a mother cell. After 3 days of growth, most filaments haddifferentiated, at least in part, to the final intracellular offspringstage and intracellular offspring could be seen in a characteristicdoublet orientation at the filament tip where the filament cell wallappeared to have lost its structure (FIG. 2F, far left column). Manyintracellular offspring of varying lengths were also no longerassociated with filaments. SEM confirmed the presence of the needle-likeholdfast structure on intracellular offspring and at the tip offilaments (FIG. 2H, b-g), and could clearly distinguish betweenundifferentiated thin and smooth filaments (FIG. 2H, a, e-g) and thosethat were broader with a heterogeneous and bulbous morphology thatcorresponded to differentiating filaments (FIG. 2H, a, g, h) (Chase etal., 1976; Ferguson et al., 1979). In addition, cell-wall remnants couldbe detected at the distal tip of differentiating filaments whereintracellular offspring had been released (FIG. 2H, h and Extended DataFIG. 1b ) (Chase et al., 1976). Together, these data demonstrate that invitro culturing of SFB supports the full differentiation of invitro-grown SFB filaments to the intracellular offspring stage andconfirms the SFB life cycle inferred from in vivo observations.

Viability and Infectivity of In Vitro Formed Intracellular Offspring

SFB were first grown on TC7 cells under aerobic conditions (FIG. 3a )until many intracellular offspring appeared. After one additional day,intracellular offspring were no longer present and the average bacteriallength was significantly longer, indicating intracellular offspringoutgrowth into filaments. Similarly, when intracellular offspring grownin vitro were separated from filaments by filtration through a 5-μmfilter and added to newly plated cells, SFB numbers increased and thenewly formed filaments differentiated into intracellular offspring atthe filament tip (FIG. 3b, c ), demonstrating filamentation anddifferentiation of in vitro-formed intracellular offspring. To assesswhether SFB grown in vitro retained their ability to colonize mice andstimulate the characteristic innate and acquired immune responses, SFBgrown in vitro for 3 days were divided into a filament/intracellularoffspring fraction and a pure intracellular offspring fraction (FIG. 5a,b ) and gavaged into germ-free mice. Colonization was firmly establishedby both inputs at 5 days after gavage (FIG. 5c ) despite a 140-foldinput difference for the filament/intracellular offspring andintracellular offspring fractions. However, unlike mice gavaged with SFBderived from faecal samples (SFB Vivo), which generally showed goodcolonization of the ileum, mice gavaged with in vitro-grown SFB (SFBVitro) had much lower numbers of SFB colonizing the ileum (FIG. 3d andExtended Data FIG. 1d ); instead they showed heavy colonization of thecaecum (FIG. 5d ). Thus, although SFB Vitro clearly can attach to theileum, these results suggest that ileal colonization may be moreefficient by intracellular offspring released from spores found in thefaecal input, possibly because of the expression of flagella at thisparticular stage of the life cycle (Pamp et al., 2012). Notably, themagnitude of the innate host response (FIG. 3e ) was proportional to thecolonization level of the ileum (FIG. 3e ) and not the overall SFBfaecal load (Extended Data FIG. 1c ), revealing the requirement forileal attachment of SFB to induce the innate host response. Similar toSFBVivo, albeit with less potency, SFB Vitro were also able to stimulatethe B-cell compartment in Peyer's patches (FIG. 3f, g ), enhance IgAsecretion in the faeces (FIG. 3h ), and increase the number of Th17cells and the level of IL-17A messenger RNA in the small intestinelamina propria (FIG. 3i-k ).

SFB-host Interaction and the Host Response to SFB Growth.

Despite the apparent requirement for close contact between host cellsand SFB for efficient growth, a tight interaction was not readilyobserved. However, when the interaction was promoted by gently spinningintracellular offspring onto cells, SFB filaments were found attached tomICcl2 cells (FIG. 4A-C). This stable interaction was accompanied byactin accumulation surrounding the filament tip (FIG. 4C) and couldleave structurally intact vacant W attachment sites (FIG. 4A, c),similar to those observed in the ileum of mice (FIG. 5e ). Attachedfilaments included both undifferentiated ones and those that had reachedthe final intracellular offspring stage. Contrary to in vivo results(Tannock et al., 1984), attachment in vitro was not species specific(FIG. 4D). To assess the similarity in the host response to SFB in vitroand in vivo, it was analysed the gene expression profile of epithelialderived host factors known to be regulated by SFB colonization (Ivanovet al., 2009; Gaboriau-Routhiau et al., 2009; Goto et al., 2014; Shimaet al., 2008; Lécuyer et al., 2014). It was found that the generegulation in vitro closely recapitulated gene regulation in vivo (FIG.4E), thereby further supporting the in vitro model system. Going beyondpreviously implicated epithelial factors, it was tested severaladditional cytokines, chemokines, and host defence genes (FIG. 4F). Thedata show that SFB growth leads to a strong inflammatory host response,with the induction of pleiotropic inflammatory mediators such astumour-necrosis factor-α (TNF-α), interleukin-1-α (IL1-α), and serumamyloid A 1-3 (Saa1-3), induction of several innate host defencemechanisms (Reg3γ, iNos, and lactoferrin), and an immunologicalenvironment that is conducive for the recruitment of B cells, thetransmigration of IgA, recruitment and activation of T cells, andrecruitment of neutrophils, dendritic cells, as well as monocytes. Inagreement, it was observed that the transcript level of most immunegenes that are upregulated during co-culture with mICcl2 cells in vitrowas also increased by SFB, and not by Escherichia coli, duringcolonization experiments in vivo (FIG. 5f ). Conversely, thetranscriptional response in the human TC7 cell line was divergent fromthat observed in mICcl2 cells and less consistent with in vivo results(FIG. 4g ). Lastly, by using an array of microbe-associated molecularpatterns (MAMPs), it was demonstrated that the inflammatory response toSFB in vitro is probably shaped by the activation of TLR2 (FIG. 4h ).

Conclusion

These data demonstrate the successful culturing of SFB in vitro andprovide new insights into SFB growth requirements and the host responseto SFB challenge. These data suggest that in vivo, attachment of SFB tothe ileal surface is an important feature to elicit epithelial cellresponses, whereas in vitro, where attachment remained infrequent, theclose proximity of SFB and cells appears largely to bypass the need forattachment to deliver the stimulating signal(s). These findingshighlight the importance of the privileged location of the replicativeniche of SFB at the ileal epithelial surface in mediating thestimulatory potential of SFB.

EXAMPLE 2 Methods for Genetically Modifying a SFB Strain

A series of modular shuttle vectors designed for use in Clostridiaspecies (Heap et al., 2009) are tested for their ability to be stablyintroduced into SFB during in vitro growth using both conjugation andtransformation. Each plasmid contains a different origin of replicationwith a choice of antibiotic resistance cassettes, multiple cloning sitesand promoters. Conjugation and transformation are performed during invitro growth of SFB.

Conjugation

An auxotrophic E. coli strain requiring the addition of Diaminopimelicacid to the medium for growth is used as a donor strain as this straincan be easily selected against in rich medium with the absence ofdiaminopimelic acid.

SFB strain is purified from monoassociated mice as described above andintracellular offsprings (IOs) are added to host cells (TC7 cells) grownon either tissue culture or transwell plates under conditions describedfor SFB growth above.

After substantial growth has occurred (2-4 days), diaminopimelic acid(100 ug/ml) and the donor E. coli strain (requiring dap addition forgrowth and carrying a plasmid) is added and centrifuged together withcells and SFB and maintained for a given time (e.g., 4-8 hours).

Alternatively, in vitro grown (cultured) SFB are collected and mixedwith the aforementioned E. coli strain and pelleted (all under lowoxygen) and maintained in a pellet for varying amounts of time (e.g.,4-8 hours).

Bacteria are collected and washed 2× with regular SFB growth mediumlacking diaminopimelic acid before being put on fresh eukaryotic cells(always at low oxygen) with regular SFB medium but complemented with theappropriate antibiotic to which the conjugal plasmid gives SFBresistance to.

After a given time (0-24 hours), SFV are collected and gavaged intogermfree mice.

Recombinant SFB are selected for and maintained by antibiotic treatmentof the drinking water. The optimal and minimal regime of antibiotic usein the drinking water that is needed to cure SFB monoassociated micefrom SFB is determined prior and used to select for recombinant SFB.

Transformation by Electroporation

As described above, SFB are grown (cultured) in vitro for 2-4 days onTC7 cells.

SFB are collected and put on ice. Under low oxygen conditions andworking with solutions put on ice, SFB are washed 6× with 10%glycerol/pre-equilibrated water before being resuspended andconcentrated with 10% glycerol/H2O.

Purified plasmid DNA is added and SFB is electroporated (6.5 kV·cm⁻¹ to25 kV·cm⁻¹).

SFB are placed in regular SFB medium again and spun onto fresh cells.SFB are allowed to recover for 3-24 hours before selective antibiotic beadded overnight.

SFB are gavaged into GF mice.

Recombinant SFB are selected for and maintained by antibiotic treatmentof the drinking water.

Transformation by the Chemical Method: Calcium Chloride and Heat Shock

As described above, SFB are grown in vitro for 2-4 days on TC7 cells.

SFB are collected and put on ice. Under low oxygen conditions andworking with solutions put on ice, SFB are washed 5× with 0.1M calciumchloride before being resuspended and concentrated with 10%glycerol/0.1M calcium chloride.

Purified plasmid DNA is added to SFB and incubated for 30 minutes.

SFB are heat shocked at 42° C. for 30 seconds before being placed inregular SFB medium again and spun onto fresh cells. SFB are allowed torecover for 3-24 hours before selective antibiotic be added overnight.

SFB are gavaged into GF mice.

Alternatively, the above procedure may be performed at room temperatureonly without heat shock.

Recombinant SFB are selected for and maintained by antibiotic treatmentof the drinking water.

REFERENCES

-   Bolotin, A. et a, Genome Announc. 2, 1-2 (2014).-   Chappert, P. et al., Immunity 38, 1198-1210 (2013).-   Chase, D. G. & Erlandsen, S. L., J Bacteria 127, 572-583 (1976).-   Danchin A., Molec. gen. Genet. 150, 293-299 (1977).-   Davis, C. P. and Savage, D. C., Infection and Immunity 13, 180-188    (1976).-   Ericsson, A. C., et al., Comparative Medicine, 64, 90-98 (2014).-   Ferguson, D. J. & Birch-Andersen, A., Acta Pathol. Microbiol. Scand.    B 87, 247-252 (1979).-   Ferreira, P. C. D. et al., FEMS Immunol Med Microbiol. 54, 245-254    (2008).-   Ferreira, P. C. D. et al., Clinical and Vaccine Immunology 18,    1823-1833 (2011).-   Fleckenstein, J. et al., Expert Review of Vaccines 13, 631-639    (2014).-   Gaboriau-Routhiau, V. et al. Immunity 31, 677-689 (2009).-   Ghaem-Maghami, M. et al., Infect. Immun. 69, 5597-5605 (2001).-   Godon, J. J. et al., Appl. Environ. Microbiol. 63, 2802-2813 (1997).-   Goto, Y. et al., Science 345, 1254009 (2014).-   He, G. et al., Proc. Natl Acad. Sci. USA 96, 4586-4591 (1999).-   Heap, J. T., et al., J. Microbiol. Methods, 78, 79-85 (2009).-   Heine, S. J. et al., Vaccine 31, 2919-2929 (2013).-   Ivanov, I. I. et al., Cell 139, 485-498 (2009).-   Jepson, M. A. et al., Infect. Immun. 61, 4001-4004 (1993).-   Kaper, J. B. et al., Nat Rev Micro 2, 123-140 (2004).-   Klaasen, H. et al., FEMS Microbiol. 88, 165-180 (1992).-   Kriegel, M. A. et al., Proc. Natl Acad. Sci. USA 108, 11548-11553    (2011).-   Kuwahara, T. et al., DNA Res. 18, 291-303 (2011).-   Lécuyer, E. et al., Immunity 40, 608-620 (2014).-   Lee, Y. K. Proc. Natl Acad. Sci. USA 108 (Suppl 1), 4615-4622    (2011).-   Martinez-Becerra, F. J. et al., Infect. Immun. 80, 1222-1231 (2012).-   Mills, J. A. et al., Infect. Immun. 56, 2933-2941 (1988).-   Paccez J. D. et al., Vaccine 25, 4671-4680 (2007).-   Pamp, S. J. et al., Genome Res. 22, 1107-1119 (2012).-   Parsot, C. Curr. Opin. Microbiol. 12, 110-116 (2009).-   Prakash, T. et al., Cell Host Microbe 10, 273-284 (2011).-   Schnupf, P. & Sansonetti, P. J., PLoS ONE 7, e36446 (2012).-   Schnupf, P. et al., Semin. Immunol. 25, 342-351 (2013).-   Sczesnak, A. et al., Cell Host Microbe 10, 260-272 (2011).-   Shima, T. et al., FEMS Immunol. Med. Microbiol. 52, 69-77 (2008).-   Tannock, G. W. et al., Appl. Environ. Microbiol. 47, 441-442 (1984).-   Wu, H. J. et al., Immunity 32, 815-827 (2010).-   Yang, Y. et al., Nature 510, 152-156 (2014).-   Yin, Y. et al. ISME J. 7, 615-621 (2012).-   Yurkovetskiy, L. et al., Immunity 39, 400-412 (2013).

The invention claimed is:
 1. An in vitro method of culturing a segmentedfilamentous bacterium (SFB) strain, comprising co-culturing said SFBstrain with a eukaryotic host cell, wherein the culture is performed atan O₂ level of less than 5% in a rich tissue culture liquid mediumcomprising bacterial medium components and iron, and wherein theco-culture is performed until the SFB strain releases intracellularoffspring or spores.
 2. The method according to claim 1, wherein the SFBstrain is a wild-type strain or a genetically modified strain.
 3. Themethod according to claim 2, wherein the genetically modified SFB strainexpresses an antigen of a diarrheal pathogen selected from the groupconsisting of Shigella, enterotoxic Escherichia coli, and attaching andeffacing lesion (A/E)-inducing enteropathogenic Escherichia coli.
 4. Themethod according to claim 3, wherein the antigen is Shigella IpaBprotein or IpaD protein.
 5. The method according to claim 3, wherein theantigen is an enterotoxic Escherichia coli antigen selected fromcolonization factor antigen I, adhesin CfaE, a heat-labile toxin, andnontoxic B subunit of heat-labile toxin.
 6. The method according toclaim 3, wherein the antigen is an attaching and effacing lesion(A/E)-inducing enteropathogenic Escherichia coli intimin, or a fragmentencoding amino adds 363 to 808 of the 94 kDa beta-intimin antigen. 7.The method according to claim 2, wherein the SFB strain is isolated froma mammal.
 8. The method according to claim 7, wherein the SFB strain isisolated from a mammal selected from the group consisting of a human anda mouse.
 9. An in vitro method of culturing a SFB strain, comprising thesteps of: a) growing eukaryotic host cells on a solid culture medium; b)transferring the eukaryotic host cells grown in step a) into aeukaryotic host cell-SFB liquid culture medium to provide a cellculture; c) challenging the cell culture of step b) with a SFB strain;and d) co-culturing the eukaryotic host cells and the SFB strain in theliquid culture medium at an O₂ level of less than 5%, wherein theco-culture is performed until the SFB strain releases intracellularoffspring or spores.
 10. The method according to claim 9, wherein theeukaryotic host cell is an epithelial cell or a cancer cell.
 11. Themethod according to claim 9, wherein the eukaryotic host cells aregastrointestinal tract cells or carcinoma cells.
 12. The methodaccording to claim 9, wherein the eukaryotic host cells are selectedfrom the group consisting of human Caco-2 cell line cells, human TC7cell line cells, human HeLa cell line cells, mouse mICcl2 cell linecells, and mouse CMT93 cell line cells.
 13. The method according toclaim 9, wherein in step a) the eukaryotic host cells are grown at an O₂level of from 0% to 5%.
 14. The method according to claim 9, wherein instep a) the eukaryotic host cells are grown at an O₂ level of from 0.5to 3%.
 15. The method according to claim 9, wherein in step a) theeukaryotic host cells are grown at an O₂ level of from 1 to 2.5%. 16.The method according to claim 9, wherein in step b) the eukaryotic hostcell-SFB liquid culture medium is a rich tissue culture mediumcomprising bacterial medium components and iron.
 17. The methodaccording to claim 16, wherein the liquid culture medium comprisesbrain-heart infusion and a yeast/peptone/casein amino-acid mixture. 18.The method according to claim 16, wherein the liquid culture medium issupplemented with from 1% to 5% decomplemented fetal calf serum.
 19. Themethod according to claim 16, wherein the liquid culture medium issupplemented with from 1% to 3% decomplemented fetal calf serum.
 20. Themethod according to claim 16, wherein the liquid culture medium issupplemented with 2% decomplemented fetal calf serum.
 21. The methodaccording to claim 16, wherein the liquid culture medium furthercomprises sugars, retinoic acid and/or nucleotides.
 22. The methodaccording to claim 16, wherein the iron is in the form of Fe²⁺/Fe³⁺,3×Fe²⁺, 3×Fe²⁺ or hemin.
 23. The method according to claim 16, whereinthe iron concentration is from 0.015 to 0.05 mM.
 24. The methodaccording to claim 16, wherein the iron concentration is from 0.02 to0.04 mM.
 25. The method according to claim 9, wherein the liquid culturemedium comprises DMEM/F12 advanced medium, supplemented with fetal calfserum, non-essential amino acids,4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, brain-heartinfusion, a yeast/peptone/casein amino-acid mixture and iron.
 26. Themethod according to claim 9, wherein in step c) the SFB strain is in theform of a filament or an intracellular offspring.
 27. The methodaccording to claim 9, wherein in step c), the SFB strain is directlycontacted with the eukaryotic host cells.
 28. The method according toclaim 27, wherein the co-culture of the eukaryotic host cells and theSFB strain is performed at an O₂ level of from 0.5 to 3%.
 29. The methodaccording to claim 9, wherein the co-culture of the eukaryotic hostcells and the SFB strain is performed at an O₂ level of from 0% to 5%.30. The method according to claim 9, wherein the co-culture of theeukaryotic host cell and the SFB is performed at an O₂ level of from 1to 2.5%.
 31. The method according to claim 9, wherein iron is at aconcentration of from 0.015 to 0.05 mM.
 32. The method according toclaim 9, further comprising recovering the cultured SFB strain, whereinthe cultured SFB strain is recovered in a form of at least one of afilament, an intracellular offspring and a spore.
 33. The methodaccording to claim 9, wherein the SFB strain is a wild-type strain or agenetically modified strain.
 34. The method of claim 9, wherein theeukaryotic host cells grown in step a) are grown until a cell confluenceof at least 20% is obtained.
 35. The method of claim 9, wherein theeukaryotic host cells grown in step a) are grown until a cell monolayeris obtained.
 36. The method of claim 9, wherein the eukaryotic hostcells grown in step a) are plated on a plate.
 37. The method of claim 9,wherein the eukaryotic host cells grown in step a) are plated on atissue culture well or transwell, prior to step b).
 38. The method ofclaim 9, wherein the seeding density of the eukaryotic host cells in a)is between 1×10⁴ and 6×10⁴ cells per cm².
 39. The method according toclaim 9, wherein the SFB strain is added in step c) when the culturedensity of the eukaryotic host cells is from 0.5+10⁵ to 3×10⁵ cells percm².
 40. The method according to claim 9, wherein the ratio in step c)between the eukaryotic host cells and the SFB strain is from 0.1 to 100.41. The method according to claim 40, wherein the ratio between theeukaryotic host cells and the SFB strain is from 0.3 to
 60. 42. Themethod according to claim 9, wherein the co-culture is performed for aduration selected from the group consisting of 1, 2, 3, 4, 5 and 6 days.43. The method according to claim 9, wherein d) comprises applyingconditions to induce spore formation, selected from adding an oxidativestress and short-term culture in the presence of an antibiotic.
 44. Afood product comprising the recovered SFB strain made by the method ofclaim
 32. 45. The food product according to claim 44, wherein the SFBstrain is alive.